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Food Hydrocolloids 22 (2008) 1607–1611

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Characterization of a-carrageenan solution behavior by field-flowfractionation and multiangle light scattering

Angelique Bourgoina,b, Earl Zablackisb, Janet B. Polic,�

aEssep Bio, University Catholic of Lyon, 25 rue du Plat, 6988 Lyon Cedex 02, FrancebAnalytical Science and Assay Development, Sanofi Pasteur, Discovery Drive, Swiftwater, PA 18370, USA

cProcess Development, Sanofi Pasteur, Discovery Drive, Swiftwater, PA 18370, USA

Received 13 July 2007; accepted 6 November 2007

Abstract

The salt mediated molecular conformation change of alpha (a)-carrageenan was studied in 0.1M solutions of NaCl, NaI, and KCl.Asymmetric Field-Flow Fractionation with multiangle laser light scattering (AFFF/MALLS) detection was used to determine theaverage molecular weight, radius of gyration, and hydrodynamic radius which were in turn used to calculate the molecular density. In the

presence of 0.1M NaCl, an inert salt that does not promote gelation, a-carrageenan has a denser structure compared to k-carrageenan ofa similar molecular weight. A distinct and dramatic increase in the molecular weight (factor of 2) was observed for a-carrageenan in0.1M NaI compared to 0.1M NaCl. This combined with only a slight change in the radius of gyration, suggests intermolecularinteraction to a more compact structure (e.g., coaxial helices). A similar increase in molecular weight is observed in 0.1M KCl,

accompanied with an approximate 50% increase in the radius of gyration as well as an increase in polydispersity. This may also beattributed to intermolecular interaction with helix formation (coaxial or lateral) or may be due to K+ cations interacting with naturallyoccurring residual i-carrageenan in the sample. As previously reported for other carrageenans the random coil to helix transition of

a-carrageenan appears to be stabilized by K+ cation or I� anion in an aqueous environment.r 2007 Elsevier Ltd. All rights reserved.

Keywords: a-Carrageenan; Conformation; Asymmetric field-flow fractionation; Multiangle light scattering; Solution conformation; Aggregation

1. Introduction

Carrageenans are linear sulfated galactans obtainedfrom red seaweeds (Rhodophyceae) that are commonlyused as thickeners, gelling agents, and stabilizers in thedairy and cosmetic industries. These polysaccharides maycontain up to 1000 galactose residues with various sulfateester substitutions and as a result many structures arepossible. The structures are classified into three maincommercial types based on the location of the sulfate estersand the presence or absence of the 3,6-anhydro ring(Fig. 1): kappa (k), iota (i), and lambda (l) with thedifferent types exhibiting different gelling properties due tosalt mediated dimerization and/or temperature (Marcelo,Saiz, & Pilar Tarazona, 2005; Piculell, 2006).

ee front matter r 2007 Elsevier Ltd. All rights reserved.

odhyd.2007.11.001

ing author.

ess: janet.poli@sanofipasteur.com (J.B. Poli).

The gelation mechanism of k- and i-carrageenans has beenpreviously described and investigated (Cuppo, Reynaers, &Paoletti, 2002; Mangione, Giacomazza, Bulone, Martorana,& San Biagio, 2003; Viebke, Borgstrom, Carlsson, Piculell, &Williams, 1998; Yuguchi, Thanh, Urakawa, & Kajiwara,2002); l-carrageenan appears not to gel in aqueous systems.Briefly, a random coil to helix conformational transitionproceeds with aggregation of helical segments to rigid rodbundles, eventually forming a gel network. Although there isa controversy concerning the exact intermolecular helixinteraction, Piculell (2006) has proposed that most reporteddata support a coaxial double helix conformation as opposedto lateral interaction.Both anions and cations contribute to molecular

processes associated with conformational transitions andgelation of carrageenans. For instance k-carrageenanforms a strong gel in the presence of potassium, rubidium,or cesium ions and a weaker gel with calcium ions;

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OO O

OHOH

OHOSO3- O

OH

H H HH

H

H

HH

OO O

OHOSO3

-

OHOSO3- O

O

H

H

H

H

H

HH

H

H

κ-carrageenan ι-carrageenan

OO

OHOH

H

H

H

O

OHH

O

H

OSO3-

HOSO3-

OSO3-

OO O

OHOSO3

-

OHOH O

O

H

H

H

H

H

HH

H H

λ-carrageenan α-carrageenan

Fig. 1. Chemical structure of ideal disaccharide repeat units for some carrageenans.

A. Bourgoin et al. / Food Hydrocolloids 22 (2008) 1607–16111608

i-carrageenan with alkali metal cations forms weak gels,while with alkali earths it forms transparent and rigid gels(Yuguchi, Urakawa, & Kajiwara, 2003). Cations tend tostabilize helix formation, whereas anions seem to have noinfluence, except in the case of iodide. It has been proposedthat for k-carrageenan, iodide ions stabilize helices throughbinding in interior cavities but at the same time mayprevent aggregation and gelation by creating a relativelyhydrophobic microenvironment (Grasdalen & Smidsrod,1981; Nerdal, Haugen, Knutsen, & Grasdalen, 1993).

Alpha (a)-carrageenan was first reported by Zablackisand Santos (1986) and was further characterized byFalshaw et al. (1996). Studies by Nuclear MagneticResonance (NMR; Falshaw et al., 1996), infrared spectro-scopy (IR; Zablackis and Santos, 1986) and Gas Chroma-tography with Mass Spectrometry detection (GC/MS;Chiovitti et al., 1997) confirm that a-carrageenan iscomposed of alternating three linked b-D-galactopyranosyland four linked 3,6-anhydro-a-D-galactopyranosyl-2-sul-fate units. Zablackis and Santos (1986) reported thatthe sodium form of a-carrageenan did not gel, yetexhibited high viscosity, and exhibited twice the capacity ofk-carrageenan to suspend cocoa particles in milk.

The solution behavior (e.g., varying temperature or ionicstrength conditions) of many carrageenans has previouslybeen studied. Techniques used include size exclusionchromatography with multiangle light scattering detection(SEC-MALS; Marcelo et al., 2005), single angle X-rayscattering (SAXS; Thanh et al., 2002; Yuguchi et al., 2002,2003), NMR (Falshaw et al., 1996; Grasdalen & Smidsrod,1981; Nerdal et al., 1993), and asymmetric field-flowfractionation (AFFF; Viebke & Williams, 2000; Wittgren,Borgstrom, Piculell, & Wahlund, 1998).

AFFF has the advantage of fractionating large macro-molecules while avoiding the problem of polymer particlesstrongly absorbed on packing material or being totallyexcluded in an SEC column. In practice the AFFF covers asize range from 5nm up to approximately 100 mm and

molecular weights (Mw) from 10 to 1012 kDa, which ismuch higher than that of SEC. Thanh et al. (2002)determined the average Mw range for different carragee-nans between 147 and 2399 kDa and a size range of60.7–109 nm. It is anticipated that a-carrageenan willbehave in a similar manner with molecular characteristicsin this range, thus AFFF is used in this investigation formolecular characterization in solution.A comprehensive description of macromolecular struc-

ture includes molecular shape as well as coil density andflexibility. The hydrodynamic radius Rh is calculated fromthe retention by AFFF while simultaneous MALS detec-tion measures the radius of gyration Rg and molar masssubsequent to separation. The mean coil density is thencalculated as the molecular density r which is the ratio ofRg/Rh. Theoretical values for molecular densities whichcorrespond to various polymer shapes are: r ¼ 0.775 forcompact sphere, r ¼ 1.862 for flexible polymer in a goodsolvent, and r ¼ 2.659 for a rigid rod with an axial ratio of100 (Wittgren et al., 1998).

1.1. Asymmetric field flow fractionation

AFFF has previously been described by many (Wahlund,2000) and is briefly summarized here. AFFF is achromatography-like technique where the separation oc-curs in a thin channel, usually 100–500 mm thick. A sampleis injected into the channel and focused onto theaccumulation wall. Next the sample components areallowed to diffuse by Brownian motion into the channel.Each sample component obtains a characteristic steady-state equilibrium distance from the wall depending on itsdiffusion coefficient. A flow through the channel is initiatedand the elution order based on diffusion coefficient isobtained. The separation is further optimized with theapplication of a constant or variable secondary crossflowto accomplish desired resolution. Subsequent to separa-tion, molar mass and Rg is determined from the light

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Table 1

Molecular characteristics of a-carrageenan and k-carrageenan

Sample Mwavg (Da) Mw/Mn Rg (nm) Rh (nm) r (Rg/Rh) Retention factor (1/l)

k-Carrageenan in 0.1M NaCl 315,800 1.24 46 27 1.70 32.7

a-Carrageenan in 0.1M NaCl 437,900 1.30 102 37 2.70 18.8

a-Carrageenan in 0.1M KCl 688,000 1.72 155 37 4.20 18.9

a-Carrageenan in 0.1M NaI 724,800 1.22 110 40 2.70 20.3

A. Bourgoin et al. / Food Hydrocolloids 22 (2008) 1607–1611 1609

scattering data, Rh is determined from the diffusioncoefficient D of the retained macromolecule, and theretention parameter used in this study are in the strongretention range of 1/l ¼ 8.3–32.7 (Table 1) as outlined byWittgren et al. (1998).

2. Materials and methods

2.1. Sample preparation

The origin and extraction of a-carrageenan fromCatenella nipae Zanardini from Burma, MSD#32428 hasbeen described previously (Zablackis and Santos, 1986).Briefly, the seaweed sample was extracted with boilingunder mild alkaline conditions, filtered, and precipitatedwith isopropanol. Next, it was dried in a 55 1C oven, theresulting sample being fibrous. The sample has beencharacterized by wet chemical methods, GLC-MS, IR,and NMR (13C and 1H) studies (Falshaw et al., 1996;Zablackis & Santos, 1986).

The sample for AFFF analysis was prepared by addinga-carrageenan fibrous powder slowly to deionized water at90 1C and stirred for 30min. No degradation of the sampleis expected at this temperature as the original isolationincluded a high temperature aqueous extraction (standardcarrageenan extraction method). The resulting solutionwas allowed to stand for 24 h at room temperature toensure complete solubility and then was filtered through a0.45 mm syringe filter. The sample concentration wasapproximately 1.0mg/mL. k-Carrageenan (FMC BioPolymer,Philadelphia, PA, USA) was prepared in a similar mannerbut with no additional heating after the sample addition.Heating was not used because k-carrageenan dissolvedproperly in solution, no gel or high viscosity appeared.NaCl and KCl (J.T. Baker, Phillipsburg, NJ, USA) andNaI (Sigma-Aldrich, St. Louis, MO, USA) were dissolvedin deionized water (MilliQ system) at 0.1M. Albuminmonomer bovine (Sigma-Aldrich, St. Louis, MO, USA)concentration was 1mg/mL for channel thickness determi-nation. Pullulan P-100 (Mw 100 kDa, BioChemika, BuchsSG, Switzerland) of �1.0mg/mL was employed fornormalization of the MALS detector as well as a systemsuitability check for the AFFF instrumentation (Benincasa& Fratte, 2004; Wittgren et al., 1998; Wittgren & Wahlund,1997). A high degree of accuracy and reproducibility forPullulan P-100 was obtained (n ¼ 9): average molecularweight (Mwavg) ¼ 121.3 kDa (rsd 0.6%), polydispersity

(Mw/Mn) ¼ 1.02 (rsd 0.3%), Rg ¼ 11.5 nm (rsd ¼ 6.5%)and Rh ¼ 8.7 nm (rsd 6.4%).

2.2. Asymmetric field-flow fractionation

AFFF with trapezoidal channel geometry was employedwith Eclipse software for instrument control (WyattTechnology Corporation, Santa Barbara, CA, USA) withAgilent 1000 HPLC pumps and UV detector (AgilentTechnologies, Inc., Santa Clara, CA, USA). The channel,defined by a Mylar spacer, has a length of 26.5 cm andbreadths bo and bL of 1.6 and 0.6 cm, respectively. Channelthickness was determined as outlined in Litzen (1993). Inorder to determine the channel thickness, w, with highaccuracy, calibration is required using an analyte with aknown diffusion coefficient. Bovine serum albumin withD ¼ 5.6� 10�11m2s�1 in 0.1 NaCl at ambient temperaturewas used (Brownsey, Noel, Parker, & Ring, 2003). Thechannel width obtained was 340 mm, slightly less thanthe nominal width of the spacer, which was 350 mm. Thechannel volume was determined to be 1.15 cm3 and theaccumulation wall consisted of a 10 kDa regeneratedcellulose filter.

2.3. Multiangle light scattering

An 18 angle DAWN HELEOS and Optilab rEXdifferential refractometer (Wyatt Technology Corporation,Santa Barbara, CA, USA) operating at a wavelength of658 nm was calibrated with high purity toluene (Sigma-Aldrich, St.Louis, MO, USA). As stated above PullulanP-100 was used for normalization. The Optilab rEXdifferential refractometer used for online concentrationmeasurements was calibrated with NaCl. Astra V software(Wyatt Technology Corporation, Santa Barbara, CA,USA) was used for evaluations of light scattering andrefractive index data. The molar mass and size calculatedare based on Debye plots using the linear Zimm method.

3. Results and discussion

AFFF/MALS is a valuable tool for the study ofcarrageenan solution behavior in various aqueous environ-ments. Both the radius of gyration and the hydrodynamicradius can be simultaneously determined in exactly thesame solution environment and the molecular densitysubsequently calculated. Also, the study of high Mw

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Fractograms

Alpha-carrageenan

with 0.1M KCl

Alpha-carrageenan

with 0.1M NaCl

Alpha-carrageenan

with 0.1NaI

Retention time (min)

10.0 15.0 20.0 25.0 30.0 35.0 40.0

rela

tive s

cale

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Fig. 3. Representative fractograms of a-carrageenan in different 0.1M

mobile phases. Channel flow is 1mlmin�1, crossflow 0.8–0mlmin�1 in

40min. The sample concentration was 1mgml�1.

cumulative molar mass

Alpha-carrageenan

with 0.1M NaCl

Alpha-carrageenan

with 0.1NaI

Alpha-carrageenan

with 0.1M KCl

molar mass (g/mol)

2.0x105 4.0x105 6.0x105 8.0x105 1.0x106 1.2x106

cu

mu

lati

ve w

eig

ht

fracti

on

0.2

0.4

0.6

0.8

Fig. 4. Cumulative weight fraction versus molar mass for a-carrageenanin three different mobile phases.

A. Bourgoin et al. / Food Hydrocolloids 22 (2008) 1607–16111610

aggregates present is possible because of increased separa-tion range of AFFF compared to SEC, where high Mwcomponents are necessarily excluded from the populationbeing studied. Throughout this investigation good agree-ment between published and experimental values forknown carrageenan solution size and shape characteristicswere observed.

The idealized disaccharide repeat units of a-carrageenanand k-carrageenan (Fig. 1) are somewhat similar (each withone sulfate hemiester and containing the 3,6- anhydro unit)compared to i-carrageenan or l-carrageenan. Thus, similarmacromolecular conformation characteristics mightbe expected for the two carrageenans. Assuming thata-carrageenan behaves as k-carrageenan, a slightly ex-panded random coil conformation in NaCl is anticipated(Wittgren et al., 1998), and the conformation plots (Rg vs.Mw) with slopes of 0.4 and 0.5, respectively, support thisassumption (Fig. 2). Referring to Table 1, a-carrageenanhas a molecular density r of 2.70 in 0.1 NaCl whilek-carrageenan in the same ionic conditions has a lowermolecular density (1.70), approximately 1.60 times lessdense. This indicates that a-carrageenan, in the sameconditions, presents a more compact structure thank-carrageenan.

Representative fractograms of a-carrageenan in NaCl, KCl,and NaI can be found in Fig. 3. In all three mobile phases theelution envelope is quite broad; however, in KCl the peak iseven broader, indicating greater polydispersity, as is shown inTable 1. Aside from the possibility of helix stabilization inthe presence of potassium ions, this may be attributed toi-carrageenan units naturally present in the sample.

Comparing a-carrageenan solution behavior in KCl(helix stabilizing) versus NaCl, the Mwavg increased afactor of 1.57 times in KCl and the Rg increased �50%but the average Rh remained essentially the same; as aresult the molecular density increased (r ¼ 4.20). Thisincrease in Rg has been reported for k-carrageenan (Thanhet al., 2002; Wittgren et al., 1998) and is attributedto intermolecular helix interaction. The unrealistically

conformation plot

Alpha-carrageenan in 0.1M

NaCl , slope 0.5

k-carrageenan in 0.1M

NaCl, slope 0.4

molar mass (g/mol)

1.0x104 1.0x105 1.0x106

rms r

ad

ius (

nm

)

100.0

Fig. 2. Conformation plot (Rg vs. molar mass) for a-carrageenan and k-carrageenan in 0.1M NaCl.

high value of r ¼ 4.20 may be attributed to an under-estimation of Rh. Referring to Fig. 2, Rh is calculated froman average retention time derived from a very broad peak.Other researchers have cited similar occurrences (Wittgrenet al., 1998).The cumulative weight fraction versus the molar mass

for all three mobile phases are shown in Fig. 4. A largeincrease in molar mass is observed for the a-carrageenan inNaI compared to NaCl, however the overall distributionremains the same. In contrast, the distribution profile forKCl compared to either NaI or NaCl is different and showsa larger fraction of high-Mw material present in KCl inaccordance with the higher polydispersity value.

4. Conclusions

In the presence of 0.1M NaCl the molecular conforma-tion of a-carrageenan is more dense than that ofk-carrageenan and may be the reason for greater abilityto suspend cocoa particles reported by Zablackis andSantos (1986); a-carrageenan appears to form a more

ARTICLE IN PRESSA. Bourgoin et al. / Food Hydrocolloids 22 (2008) 1607–1611 1611

compact structure in the presence of potassium ions ascompared to sodium ions. Also, evidence of helixstabilization is observed in the presence of NaI. Thissuggests that potassium as well as iodide ions stabilize theformation of helices. Based on our study we cannot saywhether the intermolecular interaction of helices is coaxialor lateral in nature.

Modification of the a-carrageenan purification to includean i-carrageenase digestion of contaminating i-carrageenansequences in the sample will be the subject of futureinvestigations. Removal of i-carrageenan sequences fromthe a-carrageenan sample may result in a more accurateestimation of Rh. The present investigation showsa-carrageenan has unique solution characteristics thatshould be investigated further.

Acknowledgments

The authors wish to thank Patricia Cash, StanleyStavinski, Jonathan Haines, Christine Mizenko, SonYunRizzo, Jamin Bennicoff, and Gregory Poli for helpfultechnical discussion and advice.

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